A large number of bacterial pathogens cause disease by delivering proteins into host cells for the purposes of disarming immune defenses or establishing sites for microbial replication in tissues. Among the most important delivery systems is the multisubunit type III secretion system. The bacterial determinants involved in moving these misregulators into host cells are known, but there is almost no information regarding how the eukaryotic cell contributes to bacterial protein movement across the host cell membrane, nor how the host facilitates recognition of targets with the cell. This application is intended to address this gap, in that it represents the first systematic attempt to identify host cll proteins that collaborate to ensure efficient translocation of bacterial proteins across the plasma membrane and movement to their intended targets. The proposed studies focus on protein delivery by Yersinia pseudotuberculosis, an enteropathogenic organism that causes systemic disease after spread from the intestine. The Aims take advantage of a novel strategy developed in the course of this work, in which a Fluorescence Resonance Energy Transfer (FRET) reporter was used to measure the amount of activity in single cells after delivery of the protein YopE during bacterial encounter. Cells having shRNAs that interfere with YopE activity were isolated, and a number of human proteins involved in assembly of a bacterial channel in the host cell plasma membrane were identified. The genes identified in the screen point to a model in which multiple components of the host cell signaling apparatus and cytoskeleton are involved in supporting the formation of a translocation channel in the host cell. The Aims of the application are directed toward testing a specific three-step hypothesis: 1) In the first step, a glycosylphosphatidylinositol (GPI)-anchored protein is engaged to facilitate the insertion of the T3SS translocation channel into the host plasma membrane. 2) Coordinate with insertion, a signaling receptor controls cytoskeletal support for translocation channel assembly. 3) The receptor-derived signal collaborates with a host cell adhesion molecule, resulting in robust Rho kinase-directed cytoskeletal activity, stabilizing the translocation channel. The GPI-anchored protein identified in this study is CD59, found on most mammalian cells, while the cell surface signaling molecule is CCR5. Both are known to support microbial pore-forming activities unrelated to type III secretion. By identifying important collaborative relationships between bacterial proteins and their host cell partners, it is hoped that sites for therapeutic interventio against microbial infections can be identified. Furthermore, the data obtained are intended to single out collaborative human proteins that may allow the identification of individuals with variant sensitivities to infectious diseases.